Live Detection of Neural Progenitors and Glioblastoma Cells by an Oligothiophene Derivative.

There is an urgent need for simple and non-invasive identification of live neural stem/progenitor cells (NSPCs) in the developing and adult brain as well as in disease, such as in brain tumors, due to the potential clinical importance in prognosis, diagnosis, and treatment of diseases of the nervous system. Here, we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) stem cell-like cells and NSPCs. While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSPCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same cell population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of neural and glioma stem and progenitor cells.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl using LC-QTOF-HRMS together with synthesized references.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 µM) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the ß position of the phenylpropanoyl moiety was highly favored; ß-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

Transthyretin Binding Mode Dichotomy of Fluorescent trans-Stilbene Ligands.

The orientations of ligands bound to the transthyretin (TTR) thyroxine (T4) binding site are difficult to predict. Conflicting binding modes of resveratrol have been reported. We previously reported two resveratrol based trans-stilbene fluorescent ligands, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (SB-11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (SB-14), that bind native and misfolded protofibrillar TTR. The binding orientations of these two analogous ligands to native tetrameric TTR were predicted to be opposite. Herein we report the crystal structures of these TTR:ligand complexes. Opposite binding modes were verified but were different than predicted. The reverse binding mode (SB-14) placing the naphthalene moiety toward the opening of the binding pocket renders the fluorescent ligand pH sensitive due to changes in Lys15 amine protonation. Conversely, the forward binding mode (SB-11) placing the naphthalene inward mediates a stabilizing conformational change, allowing intersubunit H-bonding between Ser117 of different monomers across the dimer interface. Our structures of TTR complexes answer important questions in ligand design and interpretation of trans-stilbene binding modes to the TTR T4 binding site.

A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-ß-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-ß-D-Glcp-(1→6)]-α-D-Galp-(1→6)-ß-D-Galp-(1→3)-ß-D-Galp-(1→}n. We demonstrated that 1 µg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.

Synthetic resin acid derivatives selectively open the hKV 7.2/7.3 channel and prevent epileptic seizures.

OBJECTIVE: About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKV ) channel hKV 7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hKV 7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hKV 7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. METHODS: Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. RESULTS: Fourteen resin acid derivatives were tested on hKV 7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hKV 7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hKV 7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hKV 11.1, hNaV 1.5, or hCaV 1.2, or on the amplitude of hKV 11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. SIGNIFICANCE: The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

Structure Elucidation of Urinary Metabolites of Fentanyl and Five Fentanyl Analogs using LC-QTOF-MS, Hepatocyte Incubations and Synthesized Reference Standards.

Fentanyl analogs constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism, and studies utilizing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis of hepatocyte incubations and/or authentic urine samples do not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study, seven motifs (2-, 3-, 4- and ß-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogs, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC-QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogs, ß-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. ß-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/ß-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogs where nothing is known about the metabolism.

Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards.

MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for the detection of synthetic cannabinoids in humans, since they are generally extensively metabolized. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesized metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesized metabolites were incubated with human liver microsomes (HLM) and the supernatants were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the fourth most abundant metabolite. The metabolic pathways were investigated using synthesized metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg and t1/2 was 2.1 min, indicating a low metabolic stability. M15, M8, and potentially M5 are suggested as suitable urinary targets.

LC-QTOF-MS Identification of Major Urinary Cyclopropylfentanyl Metabolites Using Synthesized Standards.

Cyclopropylfentanyl is a fentanyl analog implicated in 78 deaths in Europe and over 100 deaths in the United States, but toxicological information including metabolism data about this drug is scarce. The aim of this study was to provide the exact structure of abundant and unique metabolites of cyclopropylfentanyl along with synthesis routes. In this study, metabolites were identified in 13 post-mortem urine samples using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Samples were analyzed with and without enzymatic hydrolysis, and seven potential metabolites were synthesized in-house to provide the identity of major metabolites. Cyclopropylfentanyl was detected in all samples, and the most abundant metabolite was norcyclopropylfentanyl (M1) that was detected in 12 out of 13 samples. Reference materials were synthesized (synthesis routes provided) to identify the exact structure of the major metabolites 4-hydroxyphenethyl cyclopropylfentanyl (M8), 3,4-dihydroxyphenethyl cyclopropylfentanyl (M5) and 4-hydroxy-3-methoxyphenethyl cyclopropylfentanyl (M9). These metabolites are suitable urinary markers of cyclopropylfentanyl intake as they are unique and detected in a majority of hydrolyzed urine samples. Minor metabolites included two quinone metabolites (M6 and M7), not previously reported for fentanyl analogs. Interestingly, with the exception of norcyclopropylfentanyl (M1), the metabolites appeared to be between 40% and 90% conjugated in urine. In total, 11 metabolites of cyclopropylfentanyl were identified, including most metabolites previously reported after hepatocyte incubation.

Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation.

Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125-209) comprising a small two-stranded ß-sheet and three α-helices. The N-terminal domain (residues 23-124) is intrinsically disordered. Expression of truncated PrP (residues 90-231) is sufficient to cause prion disease and residues 90/100-231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards ß-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular ß-sheet model structure of the PrP90-231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.

Phenolic Bis-styrylbenzo[ c]-1,2,5-thiadiazoles as Probes for Fluorescence Microscopy Mapping of Aß Plaque Heterogeneity.

A fluorescent bis-styryl-benzothiadiazole (BTD) with carboxylic acid functional groups (X-34/Congo red analogue) showed lower binding affinity toward Aß1-42 and Aß1-40 fibrils than its neutral analogue. Hence, variable patterns of neutral OH-substituted bis-styryl-BTDs were generated. All bis-styryl-BTDs showed higher binding affinity to Aß1-42 fibrils than to Aß1-40 fibrils. The para-OH on the phenyl rings was beneficial for binding affinity while a meta-OH decreased the affinity. Differential staining of transgenic mouse Aß amyloid plaque cores compared to peripheral coronas using neutral compared to anionic bis-styryl ligands indicate differential recognition of amyloid polymorphs. Hyperspectral imaging of transgenic mouse Aß plaque stained with uncharged para-hydroxyl substituted bis-styryl-BTD implicated differences in binding site polarity of polymorphic amyloid plaque. Most properties of the corresponding bis-styryl-BTD were retained with a rigid alkyne linker rendering a probe insensitive to cis-trans isomerization. These new BTD-based ligands are promising probes for spectral imaging of different Aß fibril polymorphs.

Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα.

Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

Intramolecular Proton and Charge Transfer of Pyrene-based trans-Stilbene Salicylic Acids Applied to Detection of Aggregated Proteins.

Two analogues to the fluorescent amyloid probe 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) were synthesized based on the trans-stilbene pyrene scaffold (Py1SA and Py2SA). The compounds show strikingly different emission spectra when bound to preformed Aß1-42 fibrils. This remarkable emission difference is retained when bound to amyloid fibrils of four distinct proteins, suggesting a common binding configuration for each molecule. Density functional theory calculations show that Py1SA is twisted, while Py2SA is more planar. Still, an analysis of the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of the two compounds indicates that the degree of electronic coupling between the pyrene and salicylic acid (SA) moieties is larger in Py1SA than in Py2SA. Excited state intramolecular proton transfer (ESIPT) coupled-charge transfer (ICT) was observed for the anionic form in polar solvents. We conclude that ICT properties of trans-stilbene derivatives can be utilized for amyloid probe design with large changes in emission spectra and decay times from analogous chemical structures depending on the detailed physical nature of the binding site.

Synthesis and Characterization of Novel Fluoro-glycosylated Porphyrins that can be Utilized as Theranostic Agents.

Invited for this month's cover picture is the group of Professor Peter Nilsson at the Department of Physics, Chemistry and Biology at Linköping University (Sweden). The cover picture shows a fluoro-glycoporphyrin that selectively targets cancer cells. The selectivity towards cancer cells are afforded due to proper functionalization of the porphyrin scaffold with specific carbohydrates, and the cancer cells can be visualized because of the intrinsic fluorescence from the porphyrin. The molecule also exhibits properties for photodynamic therapy and the incorporation of fluorine, in the form of 2-fluoro-2-deoxy glucose (FDG), offers the possibility to apply these compounds as 18F positron emission tomography (PET) tracers. Thus, fluoro-glycoporphyrins display multimodal properties and can be employed as theranostic agents targeting cancer cells. Read the full text of their Full Paper at https://doi.org/10.1002/open.201800020.

Synthesis and Characterization of Novel Fluoro-glycosylated Porphyrins that can be Utilized as Theranostic Agents.

Small molecules with modalities for a variety of imaging techniques as well as therapeutic activity are essential, as such molecules render opportunities to simultaneously conduct diagnosis and targeted therapy, so called theranostics. In this regard, glycoporphyrins have proven useful as theranostic agents towards cancer, as well as noncancerous conditions. Herein, the synthesis and characterization of heterobifunctional glycoconjugated porphyrins with two different sugar moieties, a common monosaccharide at three sites, and a 2-fluoro-2-deoxy glucose (FDG) moiety at the fourth site are presented. The fluoro-glycoconjugated porphyrins exhibit properties for multimodal imaging and photodynamic therapy, as well as specificity towards cancer cells. We foresee that our findings might aid in the chemical design of heterobifunctional glycoconjugated porphyrins that could be utilized as theranostic agents.

Atom-by-atom tuning of the electrostatic potassium-channel modulator dehydroabietic acid.

Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel's positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel's voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug.

Detection and Imaging of Aß1-42 and Tau Fibrils by Redesigned Fluorescent X-34 Analogues.

We revisited the Congo red analogue 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) to develop this highly fluorescent amyloid dye for imaging Alzheimer's disease (AD) pathology comprising Aß and Tau fibrils. A selection of ligands with distinct optical properties were synthesized by replacing the central benzene unit of X-34, with other heterocyclic moieties. Full photophysical characterization was performed, including recording absorbance and fluorescence spectra, Stokes shift, quantum yield and fluorescence lifetimes. All ligands displayed high affinity towards recombinant amyloid fibrils of Aß1-42 (13-300 nm Kd ) and Tau (16-200 nm Kd ) as well as selectivity towards the corresponding disease-associated protein aggregates in AD tissue. We observed that these ligands efficiently displaced X-34, but not Pittsburgh compound B (PiB) from recombinant Aß1-42 amyloid fibrils, arguing for retained targeting of the Congo red type binding site. We foresee that the X-34 scaffold offers the possibility to develop novel high-affinity ligands for Aß pathology found in human AD brain in a different mode compared with PiB, potentially recognizing different polymorphs of Aß fibrils.

Prediction of designer drugs: Synthesis and spectroscopic analysis of synthetic cathinone analogs that may appear on the Swedish drug market.

The use of hyphenated analytical techniques in forensic drug screening enables simultaneous identification of a wide range of different compounds. However, the appearance of drug seizures containing new substances, mainly new psychoactive substances (NPS), is steadily increasing. These new and other already known substances often possess structural similarities and consequently they exhibit spectral data with slight differences. This situation has made the criteria that ensure indubitable identification of compounds increasingly important. In this work, 6 new synthetic cathinones that have not yet appeared in any Swedish drug seizures were synthesized. Their chemical structures were similar to those of already known cathinone analogs of which 42 were also included in the study. Hence, a total of 48 synthetic cathinones making up sets of homologous and regioisomeric compounds were used to challenge the capabilities of various analytical techniques commonly applied in forensic drug screening, ie, gas chromatography-mass spectrometry (GC-MS), gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR), nuclear magnetic resonance (NMR), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Special attention was paid to the capabilities of GC-MS and GC-FTIR to distinguish between the synthetic cathinones and the results showed that neither GC-MS nor GC-FTIR alone can successfully differentiate between all synthetic cathinones. However, the 2 techniques proved to be complementary and their combined use is therefore beneficial. For example, the structural homologs were better differentiated by GC-MS, while GC-FTIR performed better for the regioisomers. Further, new spectroscopic data of the synthesized cathinone analogs is hereby presented for the forensic community. The synthetic work also showed that cathinone reference compounds can be produced in few reaction steps.

A drug pocket at the lipid bilayer-potassium channel interface.

Many pharmaceutical drugs against neurological and cardiovascular disorders exert their therapeutic effects by binding to specific sites on voltage-gated ion channels of neurons or cardiomyocytes. To date, all molecules targeting known ion channel sites bind to protein pockets that are mainly surrounded by water. We describe a lipid-protein drug-binding pocket of a potassium channel. We synthesized and electrophysiologically tested 125 derivatives, analogs, and related compounds to dehydroabietic acid. Functional data in combination with docking and molecular dynamics simulations mapped a binding site for small-molecule compounds at the interface between the lipid bilayer and the transmembrane segments S3 and S4 of the voltage-sensor domain. This fundamentally new binding site for small-molecule compounds paves the way for the design of new types of drugs against diseases caused by altered excitability.

25C-NBOMe and 25I-NBOMe metabolite studies in human hepatocytes, in vivo mouse and human urine with high-resolution mass spectrometry.

25C-NBOMe and 25I-NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I-NBOMe, whereas 25C-NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 µM 25C-NBOMe and 25I-NBOMe; samples were analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C-NBOMe and 25I-NBOMe were predominantly metabolized by O-demethylation, followed by O-di-demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C-NBOMe urine targets are 5'-desmethyl 25C-NBOMe, 25C-NBOMe and 5-hydroxy 25C-NBOMe, and for 25I-NBOMe 2' and 5'-desmethyl 25I-NBOMe and hydroxy 25I-NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.

trans-Stilbenoids with Extended Fluorescence Lifetimes for the Characterization of Amyloid Fibrils.

It was previously reported that two naphthyl-based trans-stilbene probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (1) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (3), can bind to both native transthyretin (TTR) and misfolded protofibrillar TTR at physiological concentrations, displaying distinct emission maxima bound to the different conformational states (>100 nm difference). To further explore this amyloid probe scaffold to obtain extended fluorescence lifetimes, two new analogues with expanded aromatic ring systems (anthracene and pyrene), (E)-4-(2-(anthracen-2-yl)vinyl)benzene-1,2-diol (4) and (E)-4-(2-(pyren-2-yl)vinyl)benzene-1,2-diol (5), were synthesized employing the palladium-catalyzed Mizoroki-Heck reaction. (E)-4-Styrylbenzene-1,2-diol (2), 3, 4, and 5 were investigated with respect to their photophysical properties in methanol and when bound to insulin, lysozyme, and Aß1-42 fibrils, including time-resolved fluorescence measurements. In conclusion, 4 and 5 can bind to both native and fibrillar TTR, becoming highly fluorescent. Compounds 2-5 bind specifically to insulin, lysozyme, and Aß1-42 fibrils with an apparent fluorescence intensity increase and moderate binding affinities. The average fluorescence lifetimes of the probes bound to Aß1-42 fibrils are 1.3 ns (2), 1.5 ns (3), 5.7 ns (4), and 29.8 ns (5). In summary, the variable aromatic moieties of the para-positioned trans-stilbenoid vinyl-benzene-1,2-diol with benzene, naphthalene, anthracene, and pyrene showed that the extended conjugated systems retained the amyloid targeting properties of the probes. Furthermore, both the anthracene and pyrene moieties extensively enhanced the fluorescence intensity and prolonged lifetimes. These attractive probe properties should improve amyloid detection and characterization by fluorescence-based techniques.